Reaction of pyruvate dehydrogenase

complex (PDC)
and
Krebs Cycle
In Cytosol

In
Mitochondria
Historical perspective:

1930: Elucidation of Glycolysis

Study of oxidation of glucose in muscle, addition
of Malonate inhibited the respiration (i.e. O2
uptake).

Malonate is an inhibitor of Succinate oxidation

to Fumerate

1935: Szent-Gyorgyi: demonstrated that little

amounts (catalytic amounts) of succinate,
fumerate, malate or oxaloacetate acelerated the
rate of respiration.
He also showed the sequence of inter-conversion:
Succinate --- Fumerate --- malate ---oxaloacetate.

1936: Martius & Knoop: Found the following sequence of reaction:

Citrate to cis-aconitase to Isocitrate to a Ketogluterate to succinate

1937: Krebs: Enzymatic conversion of Pyruvate + Oxaloacetate to citrate and CO2

Discovered the cycle of these reactions and found it to be a major pathway for pyruvate
oxidation in muscle.
 Reaction of pyruvate dehydrogenase complex (PDC)

Reactions of TCA cycle: 8 reactions:

Citrate synthase
Aconitase
Iso-citrate dehydrogenase
a ketoglutarate dehydrogenase
Succinyl-Coenzyme A synthetase
Succinate dehydrogenase
Fumerase
Malate dehydrogenase
Pyruvate dehydrogenase Complex (PDC)

It is a multi-enzyme complex containing three enzymes associated together

non-covalently:

E-1 : Pyruvate dehydrogenase , uses Thiamine pyrophosphate as cofactor

bound to E1

E-2 : Dihydrolipoyl transacetylase, Lipoic acid bound, CoA as substrate

E-3 : Dihydrolipoyl Dehydrogenase FAD bound, NAD+ as substrate

Advantages of multienzyme complex:

1. Higher rate of reaction: Because product of one enzyme acts as a substrate

of other, and is available for the active site of next enzyme without much
diffusion.
2. Minimum side reaction.
3. Coordinated control.
Biochemistry

Pyruvate Dehydrogenase
Glycolysis occurs in
the cytosol of cells. matrix

Pyruvate enters the inter-

mitochondrion to be cristae membrane
metabolized further. space

Mitochondrial inner outer

membrane mitochondrion membrane
Compartments:
 The matrix contains Pyruvate Dehydrogenase,
enzymes of Krebs Cycle, and other pathways, e.g.,
fatty acid oxidation & amino acid metabolism.
 The outer membrane contains large VDAC channels,
similar to bacterial porin channels, making the outer
membrane leaky to ions & small molecules.
 matrix

The inner inter-

membrane is the cristae membrane
space
major permeability
barrier of the inner outer
mitochondrion. membrane mitochondrion membrane

It contains various transport catalysts, including a carrier

protein that allows pyruvate to enter the matrix.
It is highly convoluted, with infoldings called cristae.
Embedded in the inner membrane are constituents of the
respiratory chain and ATP Synthase.
 Pyruvate Dehydrogenase
O O HSCo A O
H 3C C C O− H3C C S CoA + CO2
pyruvate acetyl-CoA
NAD+ NADH

Pyruvate Dehydrogenase, catalyzes oxidative

decarboxylation of pyruvate, to form acetyl-CoA.
Pyruvate
Dehydrogenase: a large
complex containing many
copies of each of 3
enzymes, E1, E2, & E3.

 The inner core of mammalian Pyruvate Dehydrogenase

is an icosahedral structure consisting of 60 copies of E2.
 At the periphery of the complex are:
• 30 copies of E1 (itself a tetramer with subunits a2b2).
• 12 copies of E3 (a homodimer), plus 12 copies of an
E3 binding protein that links E3 to E2.
 Pyruvate Dehydrogenase Subunits

Enzyme Abbreviated Prosthetic Group

Pyruvate E1 Thiamine
Dehydrogenase pyrophosphate (TPP)

Dihydrolipoyl E2 Lipoamide
Transacetylase

Dihydrolipoyl E3 FAD
Dehydrogenase
dimethylisoalloxazine O O
H H H
C N C − + C N C
H3C C C C NH 2e +2H H3C C C C NH

H3C C C C C O H3C C C C C O
C N N C N N
H H H
CH2 CH2

HC OH HC OH

HC OH HC OH
FAD Adenine
FADH2
HC OH O O HC OH O O Adenine

H2C O P O P O Ribose H2C O P O P O Ribose

O- O- O- O-

FAD (Flavin Adenine Dinucleotide is derived from the

vitamin riboflavin. The dimethylisoalloxazine ring system
undergoes oxidation/reduction.
FAD is a prosthetic group, permanently part of E3.
Reaction: FAD + 2 e- + 2 H+ → FADH2
 O O
H3C
CH2 CH2 O P O P O−

CH2
+
N O− O−
N S
C
H acidic H+
H3C N NH2
thiamine pyrophosphate (TPP)

Thiamine pyrophosphate (TPP) is a derivative of

thiamine (vitamin B1).
Nutritional deficiency of thiamine leads to the disease
beriberi.
Beriberi affects especially the brain, because TPP is
required for CHO metabolism, & the brain depends on
glucose metabolism for energy.
 O O
H3C
CH2 CH2 O P O P O−

CH2
+
N O− O−
N S
C
H acidic H+
H3C N NH2
thiamine pyrophosphate (TPP)

Thiamine pyrophosphate (TPP) mechanism: O O−

H+ readily dissociates from the C between N C
and S in the thiazole ring. C O

The resulting carbanion (ylide) can attack the CH3

electron-deficient keto carbon of pyruvate. pyruvate
 S CH2
CH2
NH
S CH lipoic acid O lysine
CH2 CH2 CH2 CH2 C NH (CH2)4 CH

lipoamide C O
Lipoamide
includes a
2e− + 2H+
dithiol that
undergoes HS CH2

oxidation/ CH2
NH
HS CH O
reduction.
CH2 CH2 CH2 CH2 C NH (CH2)4 CH
dihydrolipoamide C O
 S CH2
CH2
NH
S CH lipoic acid O lysine
CH2 CH2 CH2 CH2 C NH (CH2)4 CH

lipoamide C O

2e− + 2H+
The carboxyl at the end of lipoic acid's hydrocarbon chain
forms an HS CH2
amide bond to the side-chain amino group of a
lysine residue ofCHE2 2, yielding lipoamide. NH
HS CH O
A long flexible CH
arm, including hydrocarbon chains of
2 CH2 CH2 CH2 C NH (CH2)4 CH
lipoate and the lysine R-group, links each lipoamide dithiol
C O
group to one of 2 lipoate-binding domains of each E2.
Lipoate-binding domains are themselves part of a flexible
strand of E2 that extends out from the core of the complex.
 S CH2
CH2
NH
S CH lipoic acid O lysine
CH2 CH2 CH2 CH2 C NH (CH2)4 CH

lipoamide C O

2e− + 2H+
The long flexible attachment allows lipoamide functional
groups toHS CH2 between E2 active sites in the core of the
swing
complex & activeCHsites
2 of E1 & E3 in the outer
NH
shell.
HS CH O
E3 binding protein that
CH2 CH binds E3 to E2 also has attached
2 CH2 CH2 C NH (CH2)4 CH
lipoamide that can exchange of reducing equivalents with
C O
lipoamide on E2.
Diagrams in: website of the laboratory of Wim Hol
article by Milne et al. (Fig. 5)
 H2O
HS S
R' As O + R' As
HS S
R R

Organic arsenicals are potent inhibitors of lipoamide-

containing enzymes such as Pyruvate Dehydrogenase.
These highly toxic compounds react with “vicinal”
dithiols such as the functional group of lipoamide.
 O
In the overall reaction
Coenzyme A-SH + HO C CH3
acetic acid catalyzed by the Pyruvate
Dehydrogenase complex,
O the acetic acid generated
is transferred to
Coenzyme A-S C CH3 + H2O
coenzyme A.
acetyl-CoA

H O O
H H
C C
NH2 NH2
The final electron acceptor
+
is NAD+. N − + N
2e + H
R R
NAD+ NADH
Sequence of reactions catalyzed by Pyruvate
Dehydrogenase complex:
1. The keto C of pyruvate reacts with the carbanion of
TPP on E1 to yield an addition compound.
The electron-pulling (+) charged N of the thiazole ring
promotes CO2 loss. Hydroxyethyl-TPP remains.
2. The hydroxyethyl carbanion on TPP of E1 reacts with
the disulfide of lipoamide on E2. What was the keto C
of pyruvate is oxidized to a carboxylic acid, as the
lipoamide disulfide is reduced to a dithiol.
The acetate formed by oxidation of the hydroxyethyl
is linked to one of the thiols of the reduced lipoamide
as a thioester (~).
Sequence of reactions (continued)
3. Acetate is transferred from the thiol of lipoamide
to the thiol of coenzyme A, yielding acetyl CoA.
4. The reduced lipoamide, swings over to the E3
active site.
Dihydrolipoamide is reoxidized to the disulfide, as
2 e− + 2 H+ are transferred to a disulfide on E3
(disulfide interchange).
5. The dithiol on E3 is reoxidized as 2 e- + 2 H+ are
transferred to FAD.
The resulting FADH2 is reoxidized by electron
transfer to NAD+, to yield NADH + H+.
Krebs Cycle
Acetyl CoA, a product of the Pyruvate Dehydrogenase
reaction, is a central compound in metabolism.
The "high energy" thioester linkage makes it an
excellent donor of the acetate moiety.

H3C C S CoA

acetyl-coenzyme A
 glucose-6-P
Glycolysis
pyruvate
fatty acids
acetyl CoA ketone bodies
cholesterol
oxaloacetate citrate

Krebs Cycle

Acetyl CoA functions as:

 input to Krebs Cycle, where the acetate moiety is
further degraded to CO2.
 donor of acetate for synthesis of fatty acids, ketone
bodies, & cholesterol.
Regulation of Pyruvate Dehydrogenase Complex:
Product inhibition by NADH & acetyl CoA:
 NADH competes with NAD+ for binding to E3.
 Acetyl CoA competes with CoA for binding to E2.
Regulation by E1 phosphorylation/dephosphorylation:
Specific regulatory Kinases & Phosphatases associated with
Pyruvate Dehydrogenase in the mitochondrial matrix:
 Pyruvate Dehydrogenase Kinases catalyze
phosphorylation of serine residues of E1, inhibiting the
complex.
 Pyruvate Dehydrogenase Phosphatases reverse this
inhibition.
Pyruvate Dehydrogenase Kinases are activated by NADH &
acetyl-CoA, providing another way the 2 major products of
Pyruvate Dehydrogenase reaction inhibit the complex.
Kinase activation involves interaction with E2 subunits to
sense changes in oxidation state & acetylation of lipoamide
caused by NADH & acetyl-CoA.
 During starvation:

 Pyruvate Dehydrogenase Kinase increases in

amount in most tissues, including skeletal muscle, via
increased gene transcription.
 Under the same conditions, the amount of Pyruvate
Dehydrogenase Phosphatase decreases.
The resulting inhibition of Pyruvate Dehydrogenase
prevents muscle and other tissues from catabolizing
glucose & gluconeogenesis precursors.
 Metabolism shifts toward fat utilization.
 Muscle protein breakdown to supply gluconeogenesis
precursors is minimized.
 Available glucose is spared for use by the brain.
 Pyruvate
Dehydro-
genase
in matrix
A Ca++-sensitive isoform
of the phosphatase that
removes Pi from E1 is
expressed in muscle. mitochondrion Ca++

The increased cytosolic Ca++ that occurs during

activation of muscle contraction can lead to Ca++ uptake
by mitochondria.
The higher Ca++ stimulates the phosphatase, &
dephosphorylation activates Pyruvate Dehydrogenase.
Thus mitochondrial metabolism may be stimulated
during exercise.
 Reactions of Citric Acid Cycle

1. Citrate synthase: Formation of Citroyl CoA intermediate.

2. Binding of Oxaloacetate to the enzyme results in
conformational change which facilitates the binding of the next
substrate, the acetyl Coenzyme A. There is a further
conformational change which leads to formation of products.
This mechanism of reaction is referred as induced fit model.
2. Aconitase: This enzyme catalyses the isomerization reaction by removing and
then adding back the water ( H and OH ) to cis-aconitate in at different
positions. Isocitrate is consumed rapidly by the next step thus deriving the
reaction in forward direction.
3. Isocitrate dehydrogenase: There are two isoforms of this enzyme, one uses
NAD+ and other uses NADP+ as electron acceptor.
4. a-Ketoglutarate dehydrogenase: This is a complex of different enzymatic
activities similar to the pyruvate dyhdogenase complex. It has the same
mechanism of reaction with E1, E2 and E3 enzyme units. NAD+ is an electron
acceptor.
5. Succinyl CoA synthatse: Sccinyl CoA, like Acetyl CoA has a thioester bond
with very negative free energy of hydrolysis. In this reaction, the hydrolysis
of the thioester bond leads to the formation of phosphoester bond with
inorganic phosphate. This phosphate is transferred to Histidine residue of the
enzyme and this high energy, unstable phosphate is finally transferred to GDP
resulting in the generation of GTP.
6. Succinate Dehydrogenase: Oxidation of succinate to fumarate. This is the
only citric acid cycle enzyme that is tightly bound to the inner mitochondrial
membrane. It is an FAD dependent enzyme.

Malonate has similar structure to Succinate, and it competitively inhibits SDH.

7. Fumarase: Hydration of Fumarate to malate: It is a highly stereospecific
enzyme. Cis-Maleate (the cis form of fumarate is not recognized by this
enzyme.
8. L-Malate dehydrogenase: Oxidation of malate to oxaloacetate: It is an
NAD+dependent enzyme. Reaction is pulled in forward direction by the next
reaction (citrate synthase reaction) as the oxaloacetate is depleted at a very fast
rate.
Conservation of energy of oxidation in the CAC: The two carbon acetyl group
generated in PDC reaction enter the CAC, and two molecules of CO2 are
released in on cycle. Thus there is complete oxidation of two carbons during
one cycle. Although the two carbons which enter the cycle become the part of
oxaloacetate, and are released as CO2 only in the third round of the cycle. The
energy released due to this oxidation is conserved in the reduction of 3 NAD+, 1
FAD molecule and synthesis of one GTP molecule which is converted to ATP.
Efficiency of Biochemical engine in Living Systems:
Oxidation of one glucose yields 2840 kJ/mole energy
Energy obtained by biological engine: 32ATP X 30.5 kJ/Mol = 976 kJ/mol
Thus 34% efficiency is obtained if calculations are done using standard
conditions. But if concentrations in the cellular condition are taken in account,
the efficiency is close to 65%.
Anaerobic bacteria us incomplete citric acid cycle for production of biosynthetic
precursors. They do not contain a-ketoglutarate dehydrogenase.
The amphibolic nature of Citric acid cycle: This pathway is utilized for the both
catabolic reactions to generate energy as well as for anabolic reactions to
generate metabolic intermediates for biosynthesis.

If the CAC intermediate are used for synthetic reactions, they are replenished by
anaplerotic reactions in the cells (indicated by red colours).
Fig. 16.16 Glyoxalate
cycle
Regulation of CAC:
Rate controlling enzymes:
Citrate synthatase
Isocitrate dehydrogenase
a-keoglutaratedehydrogenase

Regulation of activity by:

Substrate availability
Product inhibition
Allosteric inhibition or activation by
other intermediates